Trimeric SARS-CoV-2 Spike proteins produced from CHO-cells in bioreactors are high quality antigens
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Abstract
The Spike protein of SARS-CoV-2 is essential for virus entry into human cells. In fact, most neutralizing antibodies against SARS-CoV-2 are directed against the Spike, making it the antigen of choice for use in vaccines and diagnostic tests. In the current pandemic context, global demand for Spike proteins has rapidly increased and could exceed hundreds of grams to kilograms annually. Coronavirus Spikes are large, heavily glycosylated, homotrimeric complexes, with inherent instability. Their poor manufacturability now threatens availability of these proteins for vaccines and diagnostic tests. Here, we outline a scalable, GMP-compliant, chemically defined process for production of a cell secreted, stabilized form of the trimeric Spike protein. The process is chemically defined and based on clonal, suspension-CHO cell populations and on protein purification via a two-step, scalable downstream process. The trimeric conformation was confirmed using electron microscopy and HPLC analysis. Binding to susceptible cells was shown using a virus-inhibition assay. The diagnostic sensitivity and specificity for detection of serum SARS-CoV-2 specific IgG1 was investigated and found to exceed that of Spike fragments (S1 and RBD). The process described here will enable production of sufficient high-quality trimeric Spike protein to meet the global demand for SARS-CoV-2 vaccines and diagnostic tests.
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SciScore for 10.1101/2020.11.15.382044: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Supernatants of high-density transient transfections under suspension culture were harvested after 10 days, mimicking a fed-batch process with ExcellGene’s animal component-free and protein-free CHO4Tx® PM (production medium) in 50 mL TubeSpin® bioreactor 50 tubes (TPP, Trasadingen, Switzerland). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Supernatants Recombinant CHO cell lines: After transfections, suspension cells were stringently selected with puromycin. CHOsuggested: CLS Cat# …SciScore for 10.1101/2020.11.15.382044: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IACUC: Supernatants of high-density transient transfections under suspension culture were harvested after 10 days, mimicking a fed-batch process with ExcellGene’s animal component-free and protein-free CHO4Tx® PM (production medium) in 50 mL TubeSpin® bioreactor 50 tubes (TPP, Trasadingen, Switzerland). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Experimental Models: Cell Lines Sentences Resources Supernatants Recombinant CHO cell lines: After transfections, suspension cells were stringently selected with puromycin. CHOsuggested: CLS Cat# 603479/p746_CHO, RRID:CVCL_0213)This mixture was inoculated onto Vero E6 cells were inoculated with SARS-CoV-2 and at 48 hours post infection, SARS-CoV-2 infection was visualized using virus nucleocapsid antigen specific staining (red) and by a cell nucleus specific staining (blue), as described in [10]. Vero E6suggested: NoneSoftware and Algorithms Sentences Resources Contrast transfer function estimation and particle picking were performed in cisTEM [16]. cisTEMsuggested: (cisTEM, RRID:SCR_016502)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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